Cytoprotection through the use of hif hydroxylase inhibitors

ABSTRACT

The invention relates to methods for conferring cytoprotection, or for inducing a cytoprotective effect, by administering a compound that inhibits HIF hydroxylase. Compounds for use in these methods are also provided.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/476,740, filed on 6 Jun. 2003; U.S. Provisional Application Ser.No. 60/476,723, filed on 6 Jun. 2003; and U.S. Provisional ApplicationSer. No. 60/554,568, filed on 19 Mar. 2004, each of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for conferring cytoprotection, or forinducing a cytoprotective effect, by administering a compound thatinhibits HIF hydroxylase. Compounds for use in these methods are alsoprovided.

BACKGROUND

Cytoprotection refers to the ability of natural and/or therapeuticagents to protect a cell against damage and death. Cells have developedcertain adaptive mechanisms, triggered in response to stress, thatextend viability, delaying or preventing apoptosis or cell death. Inmany instances, however, natural cytoprotective mechanisms areinsufficient, inadequate, or induced too late to provide necessarybenefit, e.g., cell survival, reduced tissue and organ damage, etc. As aresult, cell death may occur by apoptotic or necrotic mechanisms.

Cell damage and cell death can result from stress conferred by variousphysiological and environmental factors. These factors can include, forexample, exposure to radiation (UV, gamma), cellular toxins and wasteproducts, environmental toxins, free radicals, and reactive oxygenspecies; hypoxia or oxygen deprivation; nutrient deprivation; growthfactor withdrawal, etc. Certain medical events and procedures, e.g.,surgical trauma, including transplantation events, etc., or varioustherapies, including radiation therapy and chemotherapy, can involveexposure of cells to various stresses and/or cytotoxic agents.Physiological conditions including infection, inflammation,malignancies, and other diseases, or events such as ischemic events, ortraumatic injury, can compromise function and viability.

Progressive damage to cells and consequently tissues and organs is acommon feature of degenerative disorders and diseases, trauma, and theprocess of aging in animals. Alterations in cell survival contribute tothe pathogenesis of numerous conditions and disorders, includinginfections, inflammation, malignancies, and other conditions; e.g.,cancer, viral and bacterial infections, autoimmune diseases,immunodeficiency disorders (e.g., AIDS, etc.), aging and associateddisorders, neurodegenerative disorders (Alzheimer's disease, Parkinson'sdisease, amyotrophic laterial sclerosis, retinitis pigmentosa,cerebellar degeneration), myelodysplastic syndromes (aplastic anemia),heart disease, cardiac injury including ischemic injury (myocardialinfarction, stroke, reperfusion injury), toxin-induced liver disease,etc.

The ability to induce and/or enhance innate cytoprotective mechanisms,to precondition against future trauma (e.g., surgery, etc.), as part ofa combinatorial therapy (e.g., to counteract some cytotoxic aspects ofan agent administered in chemotherapy, etc.), and to ameliorate theconsequences of exposure to physiological and/or environmental stresses,would be beneficial.

The present invention answers this need by providing methods forconferring cytoprotection on and for inducing or enhancingcytoprotective effects. In particular, the invention provides methodsand compositions for the protection of cells, tissues, organs, andorganisms, in vivo and in vitro.

SUMMARY OF THE INVENTION

The invention provides a method for conferring cytoprotection on a cell,the method comprising administering to the cell an effective amount of acompound that inhibits HIF hydroxylase activity. A method for inducing acytoprotective effect in a cell, the method comprising administering tothe cell an effective amount of a compound that inhibits HIF hydroxylaseactivity, is also provided. In various embodiments, the cytoprotectiveeffect is selected from the group consisting of increased energypreservation, increased anaerobic respiration, reduced oxygenconsumption, reduced oxidative damage, prevention or reduction ofapoptosis and inhibition of pro-apoptotic activities, and increasedexpression of cytoprotective factors, such as EPO and VEGF. Inparticular, the present invention provides methods and compounds for usein inducing HIF-regulated factors associated with cytoprotectiveprocesses including, e.g., angiogenic factors, modulators of apoptosis,regulators of energy consumption, anti-oxidant factors, and other cyto-and tissue-protective agents, etc.

The compounds of the invention are compounds that inhibit HIFhydroxylase activity. In one embodiment, the compound of the inventionis selected from the group consisting of phenanthrolines; heterocycliccarbonyl glycines including, but not limited to, substitutedquinoline-2-carboxamides and isoquinoline-3-carboxamides; andN-substituted arylsulfonylamino hydroxamic acids. In preferredembodiments, the compound of the invention is selected from the groupconsisting of 4-Oxo-1,4-dihydro-[1,10]phenanthroline-3-carboxylic acid(Compound A),3-{[4-(3,3-Dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(Compound B),[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound C),[(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound D),[(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound E),[(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound F),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound G), and[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound H).

The invention provides a method for reducing or preventing apoptosis ina subject, the method comprising administering to the subject aneffective amount of a compound that inhibits HIF hydroxylase activity.In one aspect, the reduction or prevention of apoptosis comprisesinducing expression of anti-apoptotic factors. In various aspects, theanti-apoptotic factor is selected from the group consisting ofadrenomedullin, heme oxygenase-1, and HSP70. Increases in expression ofthese anti-apoptotic factors can be measured by any of the methodsavailable to one of skill in the art, including, e.g., measuring geneexpression using microarray analysis, or by measuring protein expressionusing ELISA or other immunoassays, etc. The reduction or prevention ofapoptosis can be measured by, e.g., reduced annexin V immunostaining ofthe cell. In specific aspects, the compound is selected from the groupof compounds consisting of Compound C and Compound D.

In another aspect, the reduction or prevention of apoptosis comprisesdecreasing expression of pro-apoptotic factors. In one aspect, thepro-apoptotic factor is selected from the group consisting of caspase-3and caspase-7. Decreased expression of pro-apoptotic factors can bemeasured by any of the methods available to one of skill in the art,including, e.g., using commercially available assays or kits, such as acommercially available fluorometric assay, etc. The reduction orprevention of apoptosis can be measured by, e.g., reduced annexin Vimmunostaining of the cell. In one preferred aspect, the compound isCompound G.

In one aspect, a method for reducing or preventing oxidative damage in asubject, the method comprising administering to the subject an effectiveamount of a compound that inhibits HIF hydroxylase activity is provided.In one aspect, the reduction or prevention of oxidative damage comprisesinducing expression of factors having anti-oxidant activity. In variousaspects, the factor having anti-oxidant activity is selected from thegroup consisting of adrenomedullin, heme oxygenase-1, and HSP70.Increases in expression of these factors can be measured by any of themethods available to one of skill in the art, including, e.g., measuringgene expression using microarray analysis, or by measuring proteinexpression using ELISA or other immunoassays, etc. The reduction orprevention of oxidative damage can be measured by, e.g., increased cellviability, for example, in a standard model of oxidative stress. Inspecific aspects, the compound is selected from the group of compoundsconsisting of Compound C and Compound D.

The invention provides a method for increasing energy preservation in asubject, the method comprising administering to the subject an effectiveamount of a compound that inhibits HIF hydroxylase activity. Methods forincreasing energy preservation in a subject, wherein the subject has lowglucose levels, or wherein the subject has impaired oxidativerespiration, are specifically contemplated. In one embodiment, theenergy preservation is ATP preservation. ATP preservation can bemeasured, e.g., by any the methods available in the art, such as byusing standard available commercial kits, etc. In certain embodiments,the compound is selected from the group consisting of Compound A,Compound B, Compound C, Compound D, Compound E, Compound F, Compound G,and Compound H.

In any of the above methods, the compounds of the invention arecompounds that inhibit HIF hydroxylase activity. In one embodiment, thecompound of the invention is selected from the group consisting ofphenanthrolines; heterocyclic carbonyl glycines including, but notlimited to, substituted quinoline-2-carboxamides andisoquinoline-3-carboxamides; and N-substituted arylsulfonylaminohydroxamic acids. In preferred embodiments, the compound of theinvention is selected from the group consisting of4-Oxo-1,4-dihydro-[1,10]phenanthroline-3-carboxylic acid (Compound A),3-{[4-(3,3-Dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(Compound B),[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound C),[(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound D),[(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound E),[(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound F),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound G), and[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound H).

In various embodiments, the present invention provides formulations ormedicaments or pharmaceutical compositions comprising the compounds ofthe invention, and methods for the manufacture and use of suchformulations or medicaments or pharmaceutical compositions. In oneembodiment, a pharmaceutical composition is provided, wherein thepharmaceutical composition comprises a compound that inhibits HIFhydroxylase activity. In another embodiment, the invention encompasses akit that comprises at least one compound that inhibits HIF hydroxylaseactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth data showing decreased caspase activity in cellstreated with a compound of the present invention.

FIG. 2 sets forth data showing preservation of ATP levels in cellstreated with a compound of the present invention.

FIG. 3 sets forth data showing increased viability of cells treated witha compound of the present invention.

FIG. 4 sets forth data showing methods and compounds of the presentinvention decreased apoptosis in cells.

FIG. 5 sets forth data showing increased viability of cells treated witha compound of the present invention.

FIG. 6 sets forth data showing methods and compounds of the presentinvention increased heme oxygenase-1 expression.

DETAILED DESCRIPTION

The present invention relates to methods and compounds for inducing acytoprotective effect in a subject. The subject can be, e.g., a cell, apopulation of cells, a tissue, an organ, or an organism. Thecytoprotective effect can be induced, as appropriate, in vivo or invitro. It is explicitly contemplated that cytoprotection might desirablybe induced under situations in which HIF-regulated cytoprotectiveeffects would not be induced through natural mechanisms, includingconditions of normal or adequate oxygen.

The present methods and compounds provide cytoprotection to cells,tissues, and organs by inducing in coordinate fashion specificcytoprotective effects. Coordinated induction refers to the ability ofthe present methods and compounds to induce in a subject a series ofcytoprotective effects, sequentially or in parallel, that contribute tothe viability of the subject. These desirable cytoprotective effectsinclude increased energy preservation, increased anaerobic respiration,reduced oxygen consumption, reduced oxidative damage, prevention orreduction of apoptosis and inhibition of pro-apoptotic activities, andincreased expression of cytoprotective factors, such as EPO and VEGF. Inparticular, the present invention provides methods and compounds for usein inducing HIF-regulated factors associated with cytoprotectiveprocesses including, e.g., angiogenic factors, modulators of apoptosis,regulators of energy consumption, anti-oxidant factors, and other cyto-and tissue-protective agents, etc.

Hypoxia inducible factor (HIF) is a transcriptional activator thatmediates changes in gene expression in response to changes in cellularoxygen concentration. HIF is a heterodimer containing anoxygen-regulated alpha subunit (HIFα) and a constitutively expressedbeta subunit (HIFβ), also known as aryl hydrocarbon receptor nucleartransporter (ARNT). In oxygenated (normoxic) cells, HIFα subunits arerapidly degraded by a mechanism that involves ubiquitination by the vonHippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Under hypoxicconditions, HIFα is not degraded, and an active HIFα/β complex isformed.

The term “HIFα” refers to the alpha subunit of hypoxia inducible factorprotein or to a fragment thereof. HIFαmay be any human or othermammalian protein, or fragment thereof, including human HIF-1α (GenbankAccession No. Q16665), HIF-2α (Genbank Accession No. AAB41495), andHIF-3α (Genbank Accession No. AAD22668); murine HIF-1α (GenbankAccession No. Q61221), HIF-2α (Genbank Accession No. BAA20130 andAAB41496), and HIF-3α (Genbank Accession No. AAC72734); rat HIF-1α(Genbank Accession No. CAA70701), HIF-2α (Genbank Accession No.CAB96612), and HIF-3α (Genbank Accession No. CAB96611); and cow HIF-1α(Genbank Accession No. BAA78675). HIFα may also be any non-mammalianprotein or fragment thereof, including Xenopus laevis HIF-1α (GenbankAccession No. CAB96628), Drosophila melanogaster HIF-1α (GenbankAccession No. JC4851), and chicken HIF-1α (Genbank Accession No.BAA34234). HIFα gene sequences may also be obtained by routine cloningtechniques, for example by using all or part of a HIFα gene sequencedescribed above as a probe to recover and determine the sequence of aHIFα gene in another species.

Fragments of HIFα include the regions defined by human HIF-1α from aminoacid 401 to 603 (Huang et al. (1998) Proc Natl Acad Sci USA95:7987-7992), amino acid 531 to 575 (Jiang et al. (1997) J Biol Chem272:19253-19260), amino acid 556 to 575 (Tanimoto et al. (2000) EMBO J.19:4298-4309), amino acid 557 to 571 (Srinivas et al. (1999) BiochemBiophys Res Commun 260:557-561), and amino acid 556 to 575 (Ivan andKaelin (2001) Science 292:464-468). Further, a fragment of HIFα includesany fragment containing at least one occurrence of the motif LXXLAP,e.g., as occurs in the HIFα native sequence at L₃₉₇TLLAP and L₅₅₉EMLAP.Additionally, a fragment of HIFα includes any fragment retaining atleast one functional or structural characteristic of HIFα.

“Amino acid sequence” or “polypeptide” as used herein, e.g., to refer toHIFα and fragments thereof, refer to an oligopeptide, peptide, orprotein sequence, or to a fragment of any of these, and to naturallyoccurring or synthetic molecules. “Fragments” can refer to any portionof a sequence that retains at least one structural or functionalcharacteristic of the protein. Immunogenic fragments or antigenicfragments refer to fragments of polypeptides, preferably, fragments ofabout five to fifteen amino acids in length, that retain at least onebiological or immunological activity. Where “amino acid sequence” isrecited to refer to the polypeptide sequence of a naturally occurringprotein molecule, “amino acid sequence” and like terms are not meant tolimit the amino acid sequence to the complete native sequence associatedwith the recited protein molecule.

The destabilization of HIFα in normoxic environments is due tohydroxylation of specific proline residues by HIF-specific prolinehydroxylases (HIF PHs). HIF-regulated genes encompass a variety offactors involved in numerous processes, including angiogenesis,erythropoiesis, glucose metabolism, and numerous cytoprotective andtissue protective mechanisms involved in producing, e.g., anti-apoptoticand anti-oxidative effects, etc. These include, e.g., glycolyticenzymes, glucose transporter (GLUT)-1, erythropoietin (EPO), andvascular endothelial growth factor (VEGF). (Jiang et al. (1996) J BiolChem 271:17771-17778; Iliopoulus et al. (1996) Proc Natl Acad Sci USA93:10595-10599; Maxwell et al. (1999) Nature 399:271-275; Sutter et al.(2000) Proc Natl Acad Sci USA 97:4748-4753; Cockman et al. (2000) J BiolChem 275:25733-25741; and Tanimoto et al. (2000) EMBO J 19:4298-4309.)

Due to its regulation of such factors, HIF has been associated withvarious cytoprotective events, including preventing or reducingapoptosis. Iron chelators, e.g., deferoxamine, at high concentrationshave been shown to protect against apoptosis induced by oxidative stressand glutathione depletion in neuronal cells, presumably due tostabilization of HIF-1, although this effect is consistent with theknown ability of chelators to diminish hydroxyl radical formation.(Zaman et al; J. Neurosci 1999 19(22):9821-9830.) Cobalt chloride, whichappears to activate HIF in corticoid cultures although it is not knownto be a HIF-PH inhibitor, also protected against oxidativestress-induced death in these cells. (Zaman, supra.)

Hypoxia-induced Akt activation protected against apoptosis in rat PC12cells subjected to serum withdrawal and chemotherapy, an effect observedalso by treatment with deferoxamine, a compound known to mimic someeffects of hypoxia. (Alvarez-Tejado et al. (2001) J Biol Chem276:22368-22374.)

It has further been noted that HIF also stimulates anti-apoptoticprotective signaling pathways mediated by Jak kinases and STATtranscription factors under hypoxic conditions. Activation of Stat5 byEPO signaling results in the production of the anti-apoptotic bcl familymember Bcl-X(L). Jak-stat pathway signaling in myocardial infarctionmodels is associated with resistance to apoptosis. (Xuan et al. (2001)Proc Natl Acad Sci USA 98:9050-9055.) Constitutive activation of theJak-Stat pathway results in high expression of the anti-apoptotic bcl2family member Bcl2 and low expression of the pro-apoptotic bcl familymember bax (Nielsen et al. (1999) Leukemia 13(5):735-738).

Therefore, it is known in the art that stabilization of HIFα underlimited hypoxic conditions correlates with protection against apoptosisin cells exposed to oxidative stress, serum withdrawal, and chemicalstress. Compounds of the invention have been shown to induce expressionof glycolytic factors, and to increase expression of various factors,e.g., VEGF and EPO, which appear to act, at least in certain contexts,in a cytoprotective capacity. Compounds of the invention have also beenshown to reduce infarct size, e.g., following myocardial infarction(data not shown). (See, e.g., International Application No. PCT/US03/38689, International Publication No. WO 03/053997, and InternationalPublication No. WO 03/049689, each of which is incorporated herein byreference in its entirety.)

The present invention establishes that compounds of the invention canfurther be used to coordinately increase expression of cytoprotectivefactors, including, e.g., anti-apoptotic factors, such as HO-1, HSP70,and adrenomedullin; to decrease expression of pro-apoptotic factors,e.g., caspase-3 and caspase-7; to increase energy preservation, e.g.,ATP preservation; to increase resistance to oxidative damage; to enhanceanaerobic respiration; and to reduce oxygen consumption. The compoundsof the present invention thus demonstrated coordinated induction ofmultiple cytoprotective effects, and successfully conferredcytoprotection as measured, e.g., by prevention of apoptosis asdemonstrated through reduced annexin V immunostaining. The compoundsspecifically reduced apoptosis in cells stressed, e.g., by variousoxidative toxins and bioactive cytokines. As provided herein, themethods and compounds of the present invention induce a coordinatedcytoprotective response that prevents apoptosis and increases ormaintains cell viability.

The compounds and methods of the present invention further demonstratethat through inhibition of HIF hydroxylase activity, aspects of thisendogenous protective response can be induced to provide cytoprotectiveeffects through the coordinated induction of multiple mechanismsincluding anti-apoptotic, anti-oxidant, and other protective factors,including those relevant to glycolytic shift and neovascularizationactivities. The present methods and compounds can be applied to achievecoordinated induction of cytoprotective effects under any conditions;including in response to a stress, to ameliorate the stress-inducedconsequences. Methods and compounds of the present invention are furtheruseful in the absence of stress under conditions in which it might bedesirable, for example, in anticipation of a stress, e.g., pretreatment,preconditioning, etc., prior to surgery, therapies, exposure to certainenvironmental conditions, etc. It is specifically contemplated that theefficacy of the present methods and compounds is not limited to efficacyunder hypoxic or impaired oxygen conditions, e.g., the present methodsand compounds can be used effectively to treat or pre-treat a subjectunder normal oxygen conditions as well under conditions in which thesubject is exposed to a stress such as hypoxia.

Methods and Compounds

Various methods are provided herein, and comprise administering to asubject a compound that inhibits HIF hydroxylase activity.

A compound of the invention is thus any compound that reduces orotherwise modulates the activity of an enzyme that hydroxylates at leastone amino acid residue on HIFα. In preferred embodiments, the compoundinhibits HIF hydroxylase activity, thereby inhibiting the hydroxylationof at least one HIFα amino acid residue, e.g., a proline residue, anasparagine residue, an arginine residue, etc. In a preferred embodiment,the residue is a proline residue. In specific embodiments, the residuecan be the P₅₆₄ residue in HIF-1α or a homologous proline in anotherHIFα isoform, or the P₄₀₂ residue in HIF-1α or a homologous proline inanother HIFα isoform, etc. In other embodiments, the present methods mayencompass inhibiting hydroxylation of at least one HIFα asparagineresidue, e.g., the N₈₀₃ residue of HIF-1α or a homologous asparagineresidue in another HIFα isoform. Compounds that can be used in themethods of the invention include, for example, iron chelators,2-oxoglutarate mimetics, and modified amino acid, e.g., proline analogs,etc.

In some embodiments, the methods and compounds of the present inventioninhibit HIF hydroyxlase activity by inhibiting the activity of at leastone 2-oxoglutarate dioxygenase family. In a preferred embodiment, theHIF hydroxylase is selected from the group consisting of EGLN-1, EGLN-2,EGLN-3, or an enzymatically active fragment thereof.

Exemplary compounds of the present invention are disclosed in, e.g.,International Publication No. WO 03/049686 and International PublicationNo. WO 03/053997, incorporated herein by reference in their entireties.Specifically, compounds of the invention include, but are not limited,for example, to phenanthrolines including those described in U.S. Pat.No. 5,916,898; U.S. Pat. No. 6,200,974; and International PublicationNo. WO 99/21860; heterocyclic carbonyl glycines including, but notlimited to, substituted quinoline-2-carboxamides and esters thereof asdescribed, e.g., in U.S. Pat. Nos. 5,719,164 and 5,726,305; substitutedisoquinoline-3-carboxamides and esters thereof as described, e.g., inU.S. Pat. No. 6,093,730; and N-substituted arylsulfonylamino hydroxamicacids as described, e.g., in International Publication No. WO 00/50390.All compounds listed in these patents, in particular, those compoundslisted in the compound claims and the final products of the workingexamples, are hereby incorporated into the present application byreference herein. Exemplary compounds from each group are4-Oxo-1,4-dihydro-[1,10]phenanthroline-3-carboxylic acid (Compound A),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound G), and3-{[4-(3,3-dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(Compound B), respectively.

Preferred compounds of the present invention include, e.g., heterocycliccarboxamides. Specifically preferred heterocyclic carboxamides include,e.g., heterocyclic carboxamides wherein the heterocycle is selected fromisoquinoline, quinoline, pyridine, cinnoline, carboline, etc. Additionalstructural classes of preferred compounds include anthraquinones,azafluorenes, azaphenanthrolines, benzimidazoles, benzofurans,benzopyrans, benzothiophenes, catechols, chromanones, α-diketones,furans, N-hydroxyamides, N-hydroxyureas, imidazoles, indazoles, indoles,isothiadiazoles, isothiazoles, isoxadiazoles, isoxazoles, α-keto acids,α-keto amides, α-keto esters, α-keto imines, oxadiazoles, oxalyl amides,oxazoles, oxazolines, purines, pyrans, ppyrazines, pyrazoles,pyrazolines, pyridazines, pyridines, quinazolines, phenanthrolines,tetrazoles, thiadiazoles, thiazoles, thiazolines, thiophenes, andtriazoles. Exemplary compounds include[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound C),[(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound D),[(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound E),[(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound F), and[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound H).

In preferred embodiments, the compounds of the invention inhibit HIFhydroxylase activity by inhibiting HIF prolyl hydroxylase activity. AHIF prolyl hydroxylase or HIF-PH is any enzyme that is capable ofhydroxylating a proline residue in the HIF protein. Preferably, theproline residue hydroxylated by HIF-PH includes the proline found withinthe motif LXXLAP, e.g., as occurs in the human HIF-1α native sequence atL₃₉₇TLLAP and L₅₅₉EMLAP. HIF-PH includes members of the Egl-Nine (EGLN)gene family described by Taylor (2001, Gene 275:125-132), andcharacterized by Aravind and Koonin (2001, Genome Biol 2:RESEARCH0007),Epstein et al. (2001, Cell 107:43-54), and Bruick and McKnight (2001,Science 294:1337-1340). Examples of HIF-PH enzymes include human SM-20(EGLN1) (GenBank Accession No. AAG33965; Dupuy et al. (2000) Genomics69:348-54), EGLN2 isoform 1 (GenBank Accession No. CAC42510; Taylor,supra), EGLN2 isoform 2 (GenBank Accession No. NP_(—)060025), and EGLN3(GenBank Accession No. CAC42511; Taylor, supra); mouse EGLN1 (GenBankAccession No. CAC42515), EGLN2 (GenBank Accession No. CAC42511), andEGLN3 (SM-20) (GenBank Accession No. CAC42517); and rat SM-20 (GenBankAccession No. AAA19321). Additionally, HIF-PH may include Caenorhabditiselegans EGL-9 (GenBank Accession No. AAD56365) and Drosophilamelanogaster CG1114 gene product (GenBank Accession No. AAF52050).HIF-PH also includes any active fragment of the foregoing full-lengthproteins.

Methods for identifying compounds of the invention are also provided. Incertain aspects, a compound of the invention is one that inhibits HIFhydroxylase activity. Assays for hydroxylase activity are standard inthe art. Such assays can directly or indirectly measure hydroxylaseactivity. For example, an assay can measure hydroxylated residues, e.g.,proline, asparagine, etc., present in the enzyme substrate, e.g., atarget protein, a synthetic peptide mimetic, or a fragment thereof.(See, e.g., Palmerini et al. (1985) J Chromatogr 339:285-292.) Areduction in hydroxylated residue, e.g., proline or asparagine, in thepresence of a compound is indicative of a compound that inhibitshydroxylase activity. Alternatively, assays can measure other productsof the hydroxylation reaction, e.g., formation of succinate from2-oxoglutarate. (See, e.g., Cunliffe et al. (1986) Biochem J240:617-619.) Kaule and Gunzler (1990; Anal Biochem 184:291-297)describe an exemplary procedure that measures production of succinatefrom 2-oxoglutarate.

Procedures such as those described above can be used to identifycompounds that modulate HIF hydroxylase activity. Target protein mayinclude HIFα or a fragment thereof, e.g., HIF(556-575). Enzyme mayinclude a HIF prolyl hydroxylase (e.g., GenBank Accession No. AAG33965,etc.), or a HIF asparaginyl hydroxylase (e.g., GenBank Accession No.AAL27308, etc.), etc., or an active fragment thereof, obtained from anysource. Enzyme may also be present in a crude cell lysate or in apartially purified form. For example, procedures that measure HIFhydroxylase activity are described in Ivan et al. (2001, Science292:464-468; and 2002, Proc Natl Acad Sci USA 99:13459-13464) andHirsila et al. (2003, J Biol Chem 278:30772-30780); additional methodsare described in International Publication No. WO 03/049686. Measuringand comparing enzyme activity in the absence and presence of thecompound will identify compounds that inhibit hydroxylation of HIFα.

A compound of the invention is one that confers cytoprotection asmeasured, for example, by reduced annnexin V staining. In certainaspects, a compound of the invention produces a measurable effect, asmeasured in vitro or in vivo, as demonstrated by a measurable indicationof induction of a cytoprotective effect. This can include, for example,a demonstrated increase in expression of cytoprotective factors, e.g.,adrenomedullin, caspace 3, caspace 7, HO-1, HSP-70, VEGF, EPO, variousglycolytic factors, etc. Such measurements can be assayed, e.g., usingmethods available in the art and those described herein by way ofexample.

Pharmaceutical Formulations and Routes of Administration

The compositions of the present invention can be delivered directly orin pharmaceutical compositions containing excipients, as is well knownin the art. Present methods of treatment can comprise administration ofan effective amount of a compound of the present invention to a subject.In various embodiments, the subject is a cell, a population of cells, atissue, an organ, or an organism. In certain embodiments, the subject isan animal, a mammal, and, most preferably, a human subject.

An effective amount, e.g., dose, of compound or drug can readily bedetermined by routine experimentation, as can an effective andconvenient route of administration and an appropriate formulation.Various formulations and drug delivery systems are available in the art.(See, e.g., Gennaro, ed. (2000) Remington's Pharmaceutical Sciences,supra; and Hardman, Limbird, and Gilman, eds. (2001) The PharmacologicalBasis of Therapeutics, supra.)

Suitable routes of administration may, for example, include oral,rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteraladministration. Primary routes for parenteral administration includeintravenous, intramuscular, and subcutaneous administration. Secondaryroutes of administration include intraperitoneal, intra-arterial,intra-articular, intracardiac, intracisternal, intradermal,intralesional, intraocular, intrapleural, intrathecal, intrauterine, andintraventricular administration. The indication to be treated, alongwith the physical, chemical, and biological properties of the drug,dictate the type of formulation and the route of administration to beused, as well as whether local or systemic delivery would be preferred.

Pharmaceutical dosage forms of a compound of the invention may beprovided in an instant release, controlled release, sustained release,or target drug-delivery system. Commonly used dosage forms include, forexample, solutions and suspensions, (micro-) emulsions, ointments, gelsand patches, liposomes, tablets, dragees, soft or hard shell capsules,suppositories, ovules, implants, amorphous or crystalline powders,aerosols, and lyophilized formulations. Depending on route ofadministration used, special devices may be required for application oradministration of the drug, such as, for example, syringes and needles,inhalers, pumps, injection pens, applicators, or special flasks.Pharmaceutical dosage forms are often composed of the drug, anexcipient(s), and a container/closure system. One or multipleexcipients, also referred to as inactive ingredients, can be added to acompound of the invention to improve or facilitate manufacturing,stability, administration, and safety of the drug, and can provide ameans to achieve a desired drug release profile. Therefore, the type ofexcipient(s) to be added to the drug can depend on various factors, suchas, for example, the physical and chemical properties of the drug, theroute of administration, and the manufacturing procedure.Pharmaceutically acceptable excipients are available in the art, andinclude those listed in various pharmacopoeias. (See, e.g., USP, JP, EP,and BP, FDA web page (www.fda.gov), Inactive Ingredient Guide 1996, andHandbook of Pharmaceutical Additives, ed. Ash; Synapse InformationResources, Inc. 2002.)

Pharmaceutical dosage forms of a compound of the present invention maybe manufactured by any of the methods well-known in the art, such as,for example, by conventional mixing, sieving, dissolving, melting,granulating, dragee-making, tabletting, suspending, extruding,spray-drying, levigating, emulsifying, (nano/micro-) encapsulating,entrapping, or lyophilization processes. As noted above, thecompositions of the present invention can include one or morephysiologically acceptable inactive ingredients that facilitateprocessing of active molecules into preparations for pharmaceutical use.

Proper formulation is dependent upon the desired route ofadministration. For intravenous injection, for example, the compositionmay be formulated in aqueous solution, if necessary usingphysiologically compatible buffers, including, for example, phosphate,histidine, or citrate for adjustment of the formulation pH, and atonicity agent, such as, for example, sodium chloride or dextrose. Fortransmucosal or nasal administration, semisolid, liquid formulations, orpatches may be preferred, possibly containing penetration enhancers.Such penetrants are generally known in the art. For oral administration,the compounds can be formulated in liquid or solid dosage forms and asinstant or controlled/sustained release formulations. Suitable dosageforms for oral ingestion by a subject include tablets, pills, dragees,hard and soft shell capsules, liquids, gels, syrups, slurries,suspensions, and emulsions. The compounds may also be formulated inrectal compositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

Solid oral dosage forms can be obtained using excipients, which mayinclude, fillers, disintegrants, binders (dry and wet), dissolutionretardants, lubricants, glidants, antiadherants, cationic exchangeresins, wetting agents, antioxidants, preservatives, coloring, andflavoring agents. These excipients can be of synthetic or naturalsource. Examples of such excipients include cellulose derivatives,citric acid, dicalcium phosphate, gelatine, magnesium carbonate,magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol,polyvinyl pyrrolidone, silicates, silicium dioxide, sodium benzoate,sorbitol, starches, stearic acid or a salt thereof, sugars (i.e.dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetableoils (hydrogenated), and waxes. Ethanol and water may serve asgranulation aides. In certain instances, coating of tablets with, forexample, a taste-masking film, a stomach acid resistant film, or arelease-retarding film is desirable. Natural and synthetic polymers, incombination with colorants, sugars, and organic solvents or water, areoften used to coat tablets, resulting in dragees. When a capsule ispreferred over a tablet, the drug powder, suspension, or solutionthereof can be delivered in a compatible hard or soft shell capsule.

In one embodiment, the compounds of the present invention can beadministered topically, such as through a skin patch, a semi-solid or aliquid formulation, for example a gel, a (micro-) emulsion, an ointment,a solution, a (nano/micro)-suspension, or a foam. The penetration of thedrug into the skin and underlying tissues can be regulated, for example,using penetration enhancers; the appropriate choice and combination oflipophilic, hydrophilic, and amphiphilic excipients, including water,organic solvents, waxes, oils, synthetic and natural polymers,surfactants, emulsifiers; by pH adjustment; and use of complexingagents. Other techniques, such as iontophoresis, may be used to regulateskin penetration of a compound of the invention. Transdermal or topicaladministration would be preferred, for example, in situations in whichlocal delivery with minimal systemic exposure is desired.

For administration by inhalation, or administration to the nose, thecompounds for use according to the present invention are convenientlydelivered in the form of a solution, suspension, emulsion, or semisolidaerosol from pressurized packs, or a nebuliser, usually with the use ofa propellant, e.g., halogenated carbons dervided from methan and ethan,carbon dioxide, or any other suitable gas. For topical aerosols,hydrocarbons like butane, isobutene, and pentane are useful. In the caseof a pressurized aerosol, the appropriate dosage unit may be determinedby providing a valve to deliver a metered amount. Capsules andcartridges of, for example, gelatin, for use in an inhaler orinsufflator, may be formulated. These typically contain a powder mix ofthe compound and a suitable powder base such as lactose or starch.

Compositions formulated for parenteral administration by injection areusually sterile and, can be presented in unit dosage forms, e.g., inampoules, syringes, injection pens, or in multi-dose containers, thelatter usually containing a preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents, such as buffers, tonicityagents, viscosity enhancing agents, surfactants, suspending anddispersing agents, antioxidants, biocompatible polymers, chelatingagents, and preservatives. Depending on the injection site, the vehiclemay contain water, a synthetic or vegetable oil, and/or organicco-solvents. In certain instances, such as with a lyophilized product ora concentrate, the parenteral formulation would be reconstituted ordiluted prior to administration. Depot formulations, providingcontrolled or sustained release of a compound of the invention, mayinclude injectable suspensions of nano/micro particles or nano/micro ornon-micronized crystals. Polymers such as poly(lactic acid),poly(glycolic acid), or copolymers thereof, can serve ascontrolled/sustained release matrices, in addition to others well knownin the art. Other depot delivery systems may be presented in form ofimplants and pumps requiring incision.

Suitable carriers for intravenous injection for the molecules of theinvention are well-known in the art and include water-based solutionscontaining a base, such as, for example, sodium hydroxide, to form anionized compound, sucrose or sodium chloride as a tonicity agent, forexample, the buffer contains phosphate or histidine. Co-solvents, suchas, for example, polyethylene glycols, may be added. These water-basedsystems are effective at dissolving compounds of the invention andproduce low toxicity upon systemic administration. The proportions ofthe components of a solution system may be varied considerably, withoutdestroying solubility and toxicity characteristics. Furthermore, theidentity of the components may be varied. For example, low-toxicitysurfactants, such as polysorbates or poloxamers, may be used, as canpolyethylene glycol or other co-solvents, biocompatible polymers such aspolyvinyl pyrrolidone may be added, and other sugars and polyols maysubstitute for dextrose.

For composition useful for the present methods of treatment, atherapeutically effective dose can be estimated initially using avariety of techniques well-known in the art. Initial doses used inanimal studies may be based on effective concentrations established incell culture assays. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from animal studies andcell culture assays.

A therapeutically effective dose or amount of a compound, agent, or drugof the present invention refers to an amount or dose of the compound,agent, or drug that results in amelioration of symptoms or aprolongation of survival in a subject. Toxicity and therapeutic efficacyof such molecules can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., bydetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio LD50/ED50. Agents that exhibit hightherapeutic indices are preferred.

The effective amount or therapeutically effective amount is the amountof the compound or pharmaceutical composition that will elicit thebiological or medical response of a tissue, system, animal, or humanthat is being sought by the researcher, veterinarian, medical doctor, orother clinician, e.g., increased cell viability, decrease or preventionof apoptosis, increased expressioin of anti-apoptotic factors, decreasedexpression of pro-apoptotic factors, increased energy preservation,prevention of oxidative damage, etc.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED50 with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and/or theroute of administration utilized. The exact formulation, route ofadministration, dosage, and dosage interval should be chosen accordingto methods known in the art, in view of the specifics of a subject'scondition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to achieve thedesired effects, i.e., minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from, for example, invitro data and animal experiments. Dosages necessary to achieve the MECwill depend on individual characteristics and route of administration.In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of agent or composition administered may be dependent on avariety of factors, including the sex, age, and weight of the subjectbeing treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack, or glass and rubberstoppers such as in vials. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

EXAMPLES

The invention will be further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.These examples are provided solely to illustrate the claimed invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

The compounds of the invention are compounds that inhibit HIFhydroxylase activity. In one embodiment, the compound of the inventionis selected from the group consisting of phenanthrolines; heterocycliccarbonyl glycines including, but not limited to, substitutedquinoline-2-carboxamides and isoquinoline-3-carboxamides; andN-substituted arylsulfonylamino hydroxamic acids. In preferredembodiments, the compound of the invention is selected from the groupconsisting of 4-Oxo-1,4-dihydro-[1,10]phenanthroline-3-carboxylic acid(Compound A),3-{[4-(3,3-Dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(Compound B),[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound C),[(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound D),[(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound E),[(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound F),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound G), and[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound H).

Example 1 Increased Adrenomedullin Gene Expression

Adrenomedullin (ADM), a hypotensive peptide highly expressed in severaltissues, including adrenal medulla, cardiac ventricle, lung, and kidney,has been associated with cytoprotective effects. For example, treatmentof retinal pigment epithelial cells with ADM ameliorated ahypoxia-induced decrease in cell number. (Udono et al. (2001) InvestOphthal Vis Sci 42:1080-1086.) Compounds and methods of the presentinvention were tested for induction of adrenomedullin in various celltypes as follows.

Hep3B cells (ATCC No. HB-8064) were grown in DMEM containing 8% fetalbovine serum. Hep3B cells were seeded into 6-well culture dishes at˜500,000 cells per well. After 8 hours, the media was changed to DMEMcontaining 0.5% fetal bovine serum and the cells were incubated for anadditional 16 hours. Compound A, compound B, compound C, compound G, orcompound H was added to the cells (25 μM final concentration) and thecells were incubated for various times. Control cells were incubatedwith vehicle (DMSO) with no compound treatment. Harvested cells wereassessed for cell viability (GUAVA), or added to RNA extraction buffer(RNeasy, Qiagen) and stored at −20° C. for subsequent RNA purification.

RNA was precipitated in 0.3 M sodium acetate (pH 5.2), 50 ng/mlglycogen, and 2.5 volumes of ethanol for one hour at −20° C. Sampleswere centrifuged and pellets were washed with cold 80% ethanol, dried,and resuspend in water. Double stranded cDNA was synthesized using aT7-(dT)24 first strand primer (Affymetrix, Inc., Santa Clara Calif.) andthe SUPERSCRIPT CHOICE system (Invitrogen) according to themanufacturer's instructions. The final cDNA was extracted with an equalvolume of 25:24:1 phenol:chloroform:isoamyl alcohol using a PHASE LOCKGEL insert (Brinkman, Inc., Westbury N.Y.). The aqueous phase wascollected and cDNA was precipitated using 0.5 volumes of 7.5 M ammoniumacetate and 2.5 volumes of ethanol. Alternatively, cDNA was purifiedusing the GENECHIP sample cleanup module (Affymetrix) according to themanufacturer's instructions.

Biotin-labeled cRNA was synthesized from the cDNA in an in vitrotranslation (IVT) reaction using a BIOARRAY HighYield RNA transcriptlabeling kit (Enzo Diagnostics, Inc., Farmingdale N.Y.) according to themanufacturer's instructions. Final labeled product was purified andfragmented using the GENECHIP sample cleanup module (Affymetrix)according to the manufacturer's instructions.

Hybridization cocktail was prepared by bringing 5 μg probe to 100 μl in1× hybridization buffer (100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01% Tween20), 100 μg/ml herring sperm DNA, 500 μg/ml acetylated BSA, 0.03 nMcontol oligo B2 (Affymetrix), and 1× GENECHIP eukaryotic hybridizationcontrol (Affymetrix). The cocktail was sequentially incubated at 99° C.for 5 minutes and 45° C. for 5 minutes, and then centrifuged for 5minutes. The Murine genome MOE430Aplus2 array (Affymetrix) was broughtto room temperature and then prehybridized with 1× hybridization bufferat 45° C. for 10 minutes with rotation. The buffer was then replacedwith 80 μl hybridization cocktail and the array was hybridized for 16hours at 45° C. at 60 rpm with counter balance. Following hybridization,arrays were washed once with 6×SSPE, 0.1% Tween 20, and then washed andstained using R-phycoerythrin-conjugated streptavidin (Molecular Probes,Eugene Oreg.), goat anti-streptavidin antibody (Vector Laboratories,Burlingame Calif.), and a GENECHIP Fluidics Station 400 instrument(Affymetrix) according to the manufacturer's EukGE-WS2v4 protocol(Affymetrix). Arrays were analyzed using a GENEARRAY scanner(Affymetrix) and Microarray Suite software (Affymetrix).

RNA quality was monitored by capillary electrophoresis (AgilentBioanalyzer). Hybridization cocktails were prepared as described(Affymetrix), and hybridized to Affymetrix human U133A arrays containing22,283 probe sets. The Human Genome U133A array (Affymetrix) representsall sequences in the Human Unigene database build 133 (National Centerfor Biotechnology Information, Bethesda Md.), including approximately14,500 well-characterized human genes. Array performance was analyzedwith Affymetrix MicroArray Suite (MAS) software and individual probesets were assigned “present”, “marginal, and “absent” calls according tosoftware defaults. Statistical analyses and filtered probe set listswere prepared using GeneSpring software (Silicon Genetics). Cutoffs for“expressed” probe sets used a combination of Affymetrix “P” calls andabsolute expression values derived from Genespring's intrinsic dataerror model. Data was normalized to averaged control samples.

Replicate microarrays were probed using RNA isolated from replicateexperiments conducted on different days. Data is reported as an averageof these two determinations.

Expression of the gene encoding adrenomedullin, represented on themicroarray, was specifically analyzed. Results shown in Table 1 beloware presented as fold-increase in adrenomedullin gene expression abovenon-treated control. TABLE 1 Compound Time Adrenomedullin A 6 hrs 3.582B 6 hrs 8.334 H 6 hrs 1.298 C 6 hrs 3.896 G 6 hrs 3.278

As shown above in Table 1, addition of various compounds of the presentinvention increased expression of the gene encoding adrenomedullin.Increased expression of adrenomedullin by compounds of the presentinvention was rapid, occurring within at least 6 hours after compoundaddition. Additionally, expression of the gene encoding adrenomedullinremained elevated and continued to increase over 48 hours followingcompound addition.

Adrenomedullin gene expression following compound treatment was alsoexamined in peripheral blood mononuclear cells (PBMCs). Whole humanblood was collected and processed immediately. The blood was dilutedwith an equal volume of phosphate buffered saline. FICOLL-PAQUE PLUS(Amersham Biosciences) was layered under the blood and the tubes werecentrifuged at 350×g for 12 minutes at room temperature. PBMCs formed avisible layer in the middle layer of the tube. PBMCs were carefullyremoved from the tube, diluted with 3 volumes of phosphate bufferedsaline, and pelleted by centrifugation for 5 minutes at 300×g at roomtemperature. PBMCs were cultured in DMEM containing 2.5% fetal bovineserum and treated with either 0.25% DMSO or compound G (5 μM) in 0.25%DMSO for 20 hours. The cells were then pelleted and stored at −20° C. inRLT lysis buffer (Qiagen Inc., Valencia, Calif.) containing 1%beta-mercaptoethanol. Total RNA was isolated using the RNeasy kit(Valencia, Calif.).

PBMCs treated with compound G showed greater than 3.518-fold increase inexpression of the gene encoding adrenomedullin compared to non-treatedcontrol cells.

Adrenomedullin gene expression was also examined in cardiomyocytestreated with compound and subsequently challenged with KCN, an inhibitorof oxidative glucose metabolism. H9c2 rat cardiomyocytes were culturedin 96-well tissue culture plates (approximately 20,000 cells per well)in DMEM containing 10% fetal bovine serum. Media was changed to DMEMcontaining 0.5% fetal bovine serum, and the cells were treated with 10μM compound C or compound D for 24 hours. Media was then replaced withglucose-free DMEM (Gibco/Invitrogen Cat. # 11966-025) containing 2 mMKCN (Sigma-Aldrich Cat. No. 207810). Results of adrenomedullin geneexpression, presented as fold-increase in gene expression above DMSOcontrol, are shown below in Table 2. TABLE 2 Treatment KCN Adm DMSOcontrol No 1.0 Cmpd. C No 4.8 Cmpd. D No 2.2 DMSO control Yes 0.9 Cmpd.C Yes 7.9 Cmpd. D Yes 4.2

As shown above in Table 2, treatment of cardiomyocytes with compound Cor compound D increased adrenomedullin gene expression above thatobserved in control cultures. Cells treated with KCN, also had increasedadrenomedullin gene expression following treatment with compound C orcompound D.

Taken together, these results indicated that methods and compounds ofthe present invention increased expression of adrenomedullin, a proteinassociated with anti-apoptotic and anti-oxidant effects, in variouscells. Induction of adrenomedullin by the compounds and methodsdisclosed herein demonstrate cytoprotective aspects of the presentinvention. Further, the induction of cytoprotective factors, includingadrenomedullin, in cells under stress, e.g., hypoxic stress (Udono etal., supra) or KCN-induced metabolic stress, demonstrate acytoprotective response in various cells using the present methods.

Example 2 Decreased Caspase Activity

The apoptosis-related cysteine proteases, e.g., caspase-3 and caspase-7,are directly involved in cell apoptosis. Activation of cyclin-dependentkinase (CDK)-2 through caspase-mediated cleavage of CDK inhibitors isinstrumental in the execution of apoptosis following caspase activation.(Levkau et al. (1998) Molec Cell 1:553-563.) Apoptosis, therefore, isassociated with increased levels of caspases and caspase activity. Thecytoprotective effects of the methods and compounds of the presentinvention were thus tested for their effect on caspase-mediatedapoptosis in cells as follows.

SH-SY5Y cells (human neuroblastoma cells) were plated in 96-well cultureplates at 60,000 cells per well. Following overnight incubation, cellswere washed one time with DMEM containing 1% fetal bovine serum andcultured in identical media with either vehicle control (DMSO) or 20 μMcompound G in a total volume of 200 μl per well. After 24 hours, cellswere washed with serum-free media and then incubated with DMEMcontaining 10% fetal bovine serum with either vehicle control (DMSO) or20 μM compound G in a total volume of 200 μl per well. Replicatecultures received DMEM with 1% fetal bovine serum and were cultured witheither vehicle control (DMSO) or 20 μM compound G, in the absence orpresence of 500 nM staurosporin, a kinase inhibitor that inducescellular apoptosis by a caspase-dependent mechanism. (Jacobsen et al.(1996) J Cell Biol. 133:1041-51.). After an additional 24 hourincubation, caspase activity was assayed using a caspase-3 and caspase-7fluorometric assay according to the manufacturer's instructions (Apo-ONEHomogenous Caspase 3/7 Assay, Promega, Wis.).

As shown in FIG. 1, caspase activity was increased in SH-SY5Y cells thatwere cultured with staurosporine, but significantly inhibited if cellswere pretreated with compound G prior to staurosporine treatment.Treatment of cells with DMSO or compound G showed no differences incaspase activity in the presence of 10% fetal bovine serum. The resultsshowed that treatment of cells with compound of the present inventionprior to challenge with staurosporine reduced caspase activity/levels.These results indicated that compound of the present invention reducedcaspase-mediated apoptosis and thus provided cytoprotection to thecells. The lack of any effect on caspase activity in cells notundergoing apoptosis, i.e. cells cultured in 10% fetal bovine serum,shows that compound G specifically inhibited caspase-mediated apoptosisin response to staurosporine addition, and that compound G was not adirect caspase inhibitor.

Example 3 ATP Preservation

Metabolic challenge, e.g., by inducing oxidative stress or inhibitingoxidative metabolism, compromises cell viability by rapidly depletingATP stores in metabolically active cells. In one aspect, cytoprotectionrequires adequate production and/or preservation of ATP in the cell tomeet the ongoing demands of maintaining cell structure and function. Todemonstrate the ability of the compounds and methods of the presentinvention to preserve ATP levels in challenged cells, the followingexperiment was performed.

H9c2 rat cardiomyocytes were incubated with 10 mM homocysteic acid (HCA)in the absence or presence of various concentrations of compound G asindicated for 24 hours. Cell viability was determined by measuringintracellular ATP levels. Quantitation of intracellular ATP levels wasperformed using the ViaLight Plus™ kit (Cambrex Cat. No. LT17-221)according to the manufacturers instructions.

HCA induces glutathione depletion in cells, thereby decreasing thereducing capacity of cells. Therefore, cells treated with HCA are underoxidative stress. As shown in FIG. 2, treatment of cells with compound Gas compared to vehicle control (DMSO) in the presence of HCA resulted indose-dependent increases in intracellular ATP levels. Intracellular ATPlevels were unchanged in cells not incubated with HCA (data not shown).

Phase contrast microscopy of cells treated with HCA correlated with theresults shown for intracellular ATP levels in vehicle (DMSO) andcompound G treated cells. Thus, cells exposed to HCA and subsequentlytreated with vehicle were sparse and showed a rounded morphology,indicative of low cell viability in response to oxidative stress. (SeeFIG. 3.) In contrast, cardiomyocytes treated with HCA in the presence of30 μM compound G were still confluent, appeared less rounded, andmaintained a morphology consistent with viable cardiomyoctes. Theseresults showed that compounds and methods of the present invention areuseful for maintaining cell viability under conditions of oxidativestress. Additionally, these results showed that methods and compounds ofthe present invention preserve intracellular ATP levels, useful andrequired for maintaining basal metabolic processes and cell viability.The results also indicated that methods and compounds of the presentinvention provide cytoprotection to cells under oxidative stress.

In similar experiments, H9c2 rat cardiomyocytes were pretreated witheither vehicle control (0.5% DMSO) or 20 μM of Compound A, Compound B,Compound C, Compound D, Compound E, Compound F, Compound G, or compoundH. After 24 hours, cells were washed with serum-free DMEM and subjectedto oxygen and glucose deprivation by incubating the cells inglucose-free DMEM and 2 mM KCN for 30 minutes. Intracellular ATP levelswere then determined. Table 3 below shows ATP levels (represented asrelative light units) in cells treated with various compounds of thepresent invention. TABLE 3 Compound ATP (relative light units) A 968.5 B683.5 C 3710 D 859.5 E 2043 F 1134 G 2128.3 H 947 Vehicle control (DMSO)415

Cells deprived of nutrients (i.e., glucose) and oxygen (i.e., inhibitionof oxidative respiration) showed a dramatic and rapid decrease inintracellular ATP levels. Cells treated with compound of the presentinvention prior to deprivation of nutrients and oxygen showed higherlevels of intracellular ATP than non-treated control cells. Theseresults indicated that methods and compounds of the present inventionare effective at preserving intracellular ATP levels in cells exposed tostress, such as low-glucose or decreased oxidative respiration. The dataalso suggested that treatment of cells, tissues, and organs with acompound of the present invention is effective for inducingcytoprotection or cytoprotective events prior to a condition of stress.

Example 4 Increase Heme Oxygenase-1 Gene Expression

Heme oxygenase (HO)-1 is known to exert various cytoprotectivemechanisms offering anti-apoptotic, anti-oxidant, and anti-inflammatoryeffects. The following experiment was performed to demonstrate that themethods and compounds of the present invention regulate expression ofheme oxygenase, and thereby induce its cytoprotective effects.

Rat H9c2 cardiomyocytes were treated with either vehicle control (DMSO)or with 10 μM of Compound C or Compound D. After 24 hours, cells wereharvested and RNA isolated for analysis of HO-1 gene expression bymicroarray analysis. Total RNA was isolated from cells using the RNeasykit (Qiagen), and prepared for microarray analysis as described above inExample 1. Microarray analysis was performed using the Murine GenomeMOE430Aplus2 array (Affymetrix) represents all sequences in the MurineUniGene database build 107 (National Center for BiotechnologyInformation, Bethesda Md.), including approximately 14,000well-characterized mouse genes.

As shown in FIG. 6, treatment of cells with either compound C orcompound D increased HO-1 gene expression 2- to 3-fold in cardiomyocytescompared to that observed in non-treated control cells.

In another series of experiments, H9c2 rat cardiomyocytes were treatedwith either vehicle control or with various concentrations (1 μM, 3 μM,10 μM, 30 μM, and 100 μM) of compound C or compound G. Cell lysates wereharvested and HO-1 protein levels determined by ELISA according to themanufacturer's instructions (cat # EKS-810; Stressgen, Victoria, BC,Canada). Data shown below in Table 4 represents values obtain with 100μM compound. TABLE 4 HO-1 Compound (ng/mg total cell protein) C 102.68 G28.54 control 2.86

As shown in Table 4 above, compounds of the present invention increasedexpression of HO-1 protein in cardiomyocytes in a dose-dependentfashion. Increased HO-1 proteins levels were observed in cells treatedwith various compounds of the present invention. The results indicatedthat methods and compounds of the present invention are useful forincreasing expression of HO-1 mRNA and protein in cells and tissues.Since HO-1 has been shown to be cytoprotective for cells and tissuesexposed to stress, e.g. ischemia, compounds and methods of the presentinvention provide cytoprotective effects on cells and tissues by, forexample, increasing expression of HO-1.

Example 5 Increased HSP70 Gene Expression

Similar to ADM and HO-1, the expression of heat shock protein (Hsp)70has been associated with cytoprotective effects in numerous systems.(See, e.g., Zhu et al. (2003) Arterioscler Thromb Vasc Biol 23(6):1055-1059; Mestril et al. (1994) J Clin Invest 93(2):759-756; Heads etal. (1995) J Mol Cell Cardiol 27(8):1669-1678.) To demonstrate inductionof Hsp70 using the current compounds and methods, the followingexperiments were performed.

Animal Dosing Study I

Twelve Swiss Webster male mice (30-32 g) were obtained from Simonsen,Inc (Gilroy, Calif.) and treated by oral gavage two times per day for2.5 days (5 doses) with a 4 ml/kg volume of either 0.5% carboxymethylcellulose (CMC; Sigma-Aldrich, St. Louis Mo.) (0 mg/kg/day) or 2.5%compound H (25 mg/ml in 0.5% CMC) (200 mg/kg/day). Four hours after thefinal dose, the mice were then sacrificed and approximately 150 mg ofliver and each kidney were isolated and stored in RNALATER solution(Ambion) at −20° C.

Animal Dosing Study III

To determine gene induction patterns over time, twenty four SwissWebster male mice (30-32 g) were obtained from Simonsen, Inc. andtreated by oral gavage with a 4 ml/kg volume of either 0.5%carboxymethyl cellulose (CMC; Sigma-Aldrich, St. Louis Mo.) (0 mg/kg) or1.25% compound H (25 mg/ml in 0.5% CMC) (100 mg/kg). At 4, 8, 16, 24,48, or 72 hours after the final dose, animals were anesthetized withisoflurane. The mice were then sacrificed and tissue samples of kidney,liver, brain, lung, and heart were isolated and stored in RNALATERsolution (Ambion) at −80° C. RNA isolation and gene expression analysiswere performed as described below.

RNA isolation was carried out using the following protocol. A section ofeach organ was diced, 875 μl of RLT buffer (RNEASY kit; Qiagen Inc.,Valencia Calif.) was added, and the pieces were homogenized for about 20seconds using a rotor-stator POLYTRON homogenizer (Kinematica, Inc.,Cincinnati Ohio). The homogenate was micro-centrifuged for 3 minutes topellet insoluble material, the supernatant was transferred to a new tubeand RNA was isolated using an RNEASY kit (Qiagen) according to themanufacturer's instructions. The RNA was eluted into 801 μL of water andquantitated with RIBOGREEN reagent (Molecular Probes, Eugene Oreg.). Theabsorbance at 260 and 280 nm was measured to determine RNA purity andconcentration.

Alternatively, tissue samples were diced and homogenized in TRIZOLreagent (Invitrogen Life Technologies, Carlsbad Calif.) using arotor-stator POLYTRON homogenizer (Kinematica). Homogenates were broughtto room temperature, 0.2 volumes chloroform was added, and samples weremixed vigorously. Mixtures were incubated at room temperature forseveral minutes and then were centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase was collected and 0.5 volumes of isopropanol wereadded. Samples were mixed, incubated at room temperature for 10 minutes,and centrifuged for 10 min at 12,000 g at 4° C. The supernatant wasremoved and the pellet was washed with 75% EtOH and centrifuged at 7,500g for 5 min at 4° C. The absorbance at 260 and 280 nm was measured todetermine RNA purity and concentration.

RNA was precipitated in 0.3 M sodium acetate (pH 5.2), 50 ng/mlglycogen, and 2.5 volumes of ethanol for one hour at −20° C. Sampleswere centrifuged and pellets were washed with cold 80% ethanol, dried,and resuspend in water. Double stranded cDNA was synthesized using aT7-(dT)24 first strand primer (Affymetrix, Inc., Santa Clara Calif.) andthe SUPERSCRIPT CHOICE system (Invitrogen) according to themanufacturer's instructions. The final cDNA was extracted with an equalvolume of 25:24:1 phenol:chloroform:isoamyl alcohol using a PHASE LOCKGEL insert (Brinkman, Inc., Westbury N.Y.). The aqueous phase wascollected and cDNA was precipitated using 0.5 volumes of 7.5 M ammoniumacetate and 2.5 volumes of ethanol. Alternatively, cDNA was purifiedusing the GENECHIP sample cleanup module (Affymetrix) according to themanufacturer's instructions.

Biotin-labeled cRNA was synthesized from the cDNA in an in vitrotranslation (IVT) reaction using a BIOARRAY HighYield RNA transcriptlabeling kit (Enzo Diagnostics, Inc., Farmingdale N.Y.) according to themanufacturer's instructions. Final labeled product was purified andfragmented using the GENECHIP sample cleanup module (Affymetrix)according to the manufacturer's instructions.

Hybridization cocktail was prepared by bringing 5 μg probe to 100 μl in1× hybridization buffer (100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01% Tween20), 100 μg/ml herring sperm DNA, 500 μg/ml acetylated BSA, 0.03 nMcontol oligo B2 (Affymetrix), and 1× GENECHIP eukaryotic hybridizationcontrol (Affymetrix). The cocktail was sequentially incubated at 99° C.for 5 minutes and 45° C. for 5 minutes, and then centrifuged for 5minutes. The Murine genome MOE430Aplus2 array (Affymetrix) was broughtto room temperature and then prehybridized with 1× hybridization bufferat 45° C. for 10 minutes with rotation. The buffer was then replacedwith 80 μL hybridization cocktail and the array was hybridized for 16hours at 45° C. at 60 rpm with counter balance. Following hybridization,arrays were washed once with 6×SSPE, 0.1% Tween 20, and then washed andstained using R-phycoerythrin-conjugated streptavidin (Molecular Probes,Eugene Oreg.), goat anti-streptavidin antibody (Vector Laboratories,Burlingame Calif.), and a GENECHIP Fluidics Station 400 instrument(Affymetrix) according to the manufacturer's EukGE-WS2v4 protocol(Affymetrix). Arrays were analyzed using a GENEARRAY scanner(Affymetrix) and Microarray Suite software (Affymetrix).

The Murine Genome MOE430Aplus2 array (Affymetrix) represents allsequences in the Murine UniGene database build 107 (National Center forBiotechnology Information, Bethesda Md.), including approximately 14,000well-characterized mouse genes.

As shown in Table 5 below, in vivo administration of compound H resultedin increased expression of the gene encoding HSP70-3 in mouse liver andlung. Additionally, TABLE 5 HSP70-3 mRNA Levels HSP70-3 mRNA LevelsAnimal Study Liver Lung III 1.441 3.0 I 2.77 ND

These results demonstrate the compounds and methods of the presentinvention increase Hsp70 in cells, thus eliciting the cytoprotectivebenefits thereof. Taken together, the results shown in Examples 1 to 5demonstrate a coordinated induction of cytoprotective factors using thepresent methods and compounds. Unlike single gene product cytoprotectiveeffects, the present methods provide a coordinate induction of theinnate cytoprotective factors and processes contained within a cell.Such induction, provided either before or subsequent to an initiatingstress, can provide substantial survival benefit to individual cells,and thus to tissues and organs as a whole. Specifically, these resultssuggested that methods and compounds of the present invention are usefulfor increasing expression of genes associated with cytoprotective andanti-oxidant effects.

Example 6 Reduced Apoptosis

Based on the results shown in Examples 1 to 5, demonstrating thecoordinated induction of cytoprotective factors, inhibition of apoptoticprocesses, and resulting cytoprotective effects, the effect of compoundsof the present invention on preventing or decreasing apoptosis wasexamined. Human umbilical vein endothelial cells (HUVEC) were plated inDMEM containing 0.5% fetal bovine serum that was supplemented with 1ng/ml of vascular endothelial growth factor. After overnight culture,the cells were washed with PBS and incubated for an additional 24 hoursin DMEM containing 0.5% fetal bovine serum and either vehicle control(DMSO) or 25 μM of compound G. The cell cultures were subsequentlywashed and re-cultured with DMEM containing 0.5% fetal bovine serumcontaining 20 ng/ml TNF-α for an additional 4 or 8 hours. The cells werethen harvested and immunostained with either FITC-conjugated isotypecontrol or FITC-conjugated Annexin V for identification anddetermination of cells undergoing apoptosis. (Koopman et al. (1994)Blood 84:1415-1420.) Annexin V preferentially binds negatively chargedphospholipids, like phosphatidylserine, which are associated with plasmamembrane changes in apoptotic cells. Annexin V binding allows for theidentification and quantitation of cells at early stages of apoptosis,when apoptosis occurs in the absence of DNA fragmentation, and thediscrimination between cell death associated with apoptosis or withnecrosis.

As shown in FIG. 5, TNF-α addition to HUVECs increased annexin Vimmunostaining, as measured by increased mean fluorescence intensity.This result indicated that apoptosis was induced in HUVECs in responseto TNF-α treatment. HUVECs treated with compound G 24 hours prior toaddition of TNF-α (for 4 or 8 hours) had reduced annexin Vimmunostaining compared to cells treated with TNF-α in the absence ofcompound G. (See FIG. 5.) Annexin V levels, as determined by meanfluorescence intensity, in cells treated with TNF-α and compound G wereessentially the same as that observed in control cells treated with DMSOalone. These results indicated that compounds and methods of the presentinvention prevented TNF-α induced apoptosis in HUVECs.

In replicate HUVEC cultures treated as described above, lightmicrographs were taken of cells treated with vehicle control (DMSO) andcompound G following stimulation with 20 ng/ml TNF-α for 4 and 8 hours(FIG. 6). At both time points, HUVECs treated with DMSO and stimulatedwith TNF-α exhibited a pro-apoptotic morphology, consistent with theAnnexin V immunostaining results described above. HUVECs displaying apro-apoptotic morphology were characterized by having rounded morphologyand by showing signs of detaching from the tissue culture plate. (SeeFIG. 6.) HUVECs treated with compound G prior to treatment with TNF-αexhibited a viable and normal morphology (FIG. 6), similar to thatobserved in cells not treated with TNF-α (data not shown).

Together, these results indicated that treatment of HUVECs with TNF-αinduced cell surface marker expression (i.e., Annexin V) and changes incellular morphology consistent with cells undergoing apoptosis.Treatment of cells with compound G for 24 hours prior to TNF-α treatmentinhibited the increase in Annexin V and resulted in cells maintaining aviable morphology and phenotype. Thus, methods and compounds of thepresent invention are cytoprotective to cells undergoing stressresponses that induce apoptosis, as shown here, e.g. by treatment ofHUVECs with TNF-α.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated herein by referencein their entirety.

1. A method for conferring cytoprotection on a cell, the methodcomprising administering to the cell an effective amount of a compoundthat inhibits HIF hydroxylase activity, thereby conferringcytoprotection on the cell.
 2. A method for inducing a cytoprotectiveeffect in a cell, the method comprising administering to the cell aneffective amount of a compound that inhibits HIF hydroxylase activity,thereby inducing the cytoprotective effect in the cell.
 3. The method ofany of claims 1 and 2, wherein the administering is in vitro.
 4. Themethod of any of claims 1 and 2, wherein the administering is in vivo.5. The method of claim 2, wherein the cytoprotective effect is selectedfrom the group consisting of increased energy preservation, increasedATP preservation, increased anaerobic respiration, reduced oxygenconsumption, reduced oxidative damage, increased expression of at leastone factor having anti-oxidant activity, prevention or reduction ofapoptosis, increased expression of at least one anti-apoptotic factor,decreased expression of at least one pro-apoptotic factor, and increasedexpression of at least one cytoprotective factor.
 6. The method of claim5, wherein the factor having anti-oxidant activity is selected from thegroup consisting of adrenomedullin, heme oxygenase-1, and HSP70.
 7. Themethod of claim 5, wherein the anti-apoptotic factor is selected fromthe group consisting of adrenomedullin, heme oxygenase-1, and HSP70. 8.The method of claim 5, wherein the pro-apoptotic factor is selected fromthe group consisting caspase-3 and caspase-7.
 9. The method of claim 5,wherein the cytoprotective factors are selected from the groupconsisting of erythropoietin and vascular endothelial cell growthfactor.
 10. A method for increasing adrenomedullin expression in a cell,the method comprising administering to the cell an effective amount of acompound that inhibits HIF hydroxylase activity, thereby increasingadrenomedullin expression in the cell.
 11. A method for increasing HSP70expression in a cell, the method comprising administering to the cell aneffective amount of a compound that inhibits HIF hydroxylase activity,thereby increasing HSP70 expression in the cell.
 12. A method forincreasing heme oxygenase-1 expression in a cell, the method comprisingadministering to the cell an effective amount of a compound thatinhibits HIF hydroxylase activity, thereby increasing heme oxygenase-1expression in the cell.
 13. A method for decreasing caspase expressionin a cell, the method comprising administering to the cell an effectiveamount of a compound that inhibits HIF hydroxylase activity, therebydecreasing caspase expression in the cell.
 14. The method of claim 13,wherein the caspase is selected from the group consisting of caspase-3and caspase-7.
 15. A method for preserving ATP levels in a cell, themethod comprising administering to the cell an effective amount of acompound that inhibits HIF hydroxylase activity, thereby preserving ATPlevels in the cell.
 16. A method for reducing or preventing apoptosis ina cell, the method comprising administering to the cell an effectiveamount of a compound that inhibits HIF hydroxylase activity, therebyreducing or preventing apoptosis in the cell.
 17. A method forincreasing expression of an anti-apoptotic factor in a cell, the methodcomprising administering to the cell an effective amount of a compoundthat inhibits HIF hydroxylase activity, thereby increasing expression ofthe anti-apoptotic factor in the cell.
 18. The method of claim 17,wherein the anti-apoptotic factor is selected from the group consistingof adrenomedullin, heme oxygenase-1, and HSP70.
 19. A method forincreasing expression of a factor having anti-oxidant activity in acell, the method comprising administering to the cell an effectiveamount of a compound that inhibits HIF hydroxylase activity, therebyincreasing expression of the factor having anti-oxidant activity in thecell.
 20. The method of claim 19, wherein the factor having anti-oxidantactivity is selected from the group consisting of adrenomedullin, hemeoxygenase-1, and HSP70.
 21. A method for reducing or preventingoxidative damage in a cell, the method comprising administering to thecell an effective amount of a compound that inhibits HIF hydroxylaseactivity, thereby reducing or preventing oxidative damage in the cell.22. A method for conferring cytoprotection to a cell exposed to or atrisk for exposure to stress, the method comprising administering to thecell an effective amount of a compound that inhibits HIF hydroxylaseactivity, thereby conferring cytoprotection to the cell.
 23. The methodof claim 22, wherein the stress is selected from the group consisting ofnutritional imbalance, growth factor imbalance, mechanical stress,thermal stress, reduced oxygen conditions, exposure to free radicals,hypoxia, and ischemia.
 24. The method of claim 22, wherein the stress isselected from the group consisting of exposure to a chemical agent, aninfectious agent, a toxin, a pollutant, a drug, and radiation.
 25. Themethod of claim 22, wherein the stress is associated with a conditionselected from the group consisting of an infection, an inflammation, animmunodeficiency disorder, anaphylaxis, an autoimmune disease, cancer, aneurodegenerative disorder, an aging-associated disorder, heart disease,and cardiac injury.
 26. The method of claim 25, wherein the infection isselected from the group consisting of a viral infection and a bacterialinfection.
 27. The method of claim 22, wherein the stress is associatedwith a medical procedure or treatment.
 28. The method of claim 27,wherein the medical procedure or treatment is selected from the groupconsisting of radiation therapy, chemotherapy, and surgery.
 29. Themethod according to any of the preceding claims, wherein the compound isselected from the group consisting of a phenanthroline; a heterocycliccarbonyl glycine; a quinoline-2-carboxamide; anisoquinoline-3-carboxamide; and an N-substituted arylsulfonylaminohydroxamic acid.
 30. The method according to any of the precedingclaims, wherein the compound is selected from the group consisting of4-Oxo-1,4-dihydro-[1,10]phenanthroline-3-carboxylic acid (Compound A),3-{[4-(3,3-Dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(Compound B),[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound C),[(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound D),[(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound E),[(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound F),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound G), and[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(Compound H).